The long range goal of this research is to isolate and characterize all of the components necessary for reconstitution of a hormonally regulated adenylyl cyclase system. Using the liver glucagon-sensitive adenylyl cyclase systems as a model system and by monitoring principally specific binding of labelled glucagon and adenylyl cyclase activity, we specifically propose during the next 5 years: 1) to study kinetics of receptor-adenylyl cyclase coupling in rat liver membranes; 2) to study short-time kinetics and explore modes of regulation by nucleotides; 3) to study the mechanism of action of GMP-P(NH)P (thought to be a general stimulator of adenylyl cyclases); 4) to study conditions to reverse "irreversible" activation caused by GMP-P(NH) P; 5) to study conditions for solubilization of rat liver adenylyl cyclase, to explore properties of the solubilized material and to study conditions for reconstituting particulate adenylyl cyclase responsive to hormone prior to purification of components; 6) to search for and identify factors other than nucleotides that may be present in liver homogenates and affect hormonal responsiveness either before solubilization or during reconstitution; 7) to use beef liver and rat adipose tissue adenylyl cyclase to explore whether findings with rat liver adenylyl cyclase are of general applicability; 8) to develop methods for purification of plasma membranes from beef liver in large scale and good yields, to characterize the beef liver adenylyl cyclase in comparison to the rat system, and to initiate large scale isolation of components after solubilization; and 9) to study conditions for reconstitution of a particulate system from solubilized components of the beef system restoring both hormonal and nucleotide regulation. BIBLIOGRAPHIC REFERENCES: Swartz, T.L. and Birnbaumer, L. (1977) "Reversal of Guanylyl Imidodiphosphate Activation of Adenylyl Cyclase (AC) by GTP", Fed. Proc., in press. Birnbaumer, L. and Swartz, T.L. (1977) "Guanyl Nucleotide Regulation of the Liver Glucagon-Sensitive Adenylyl Cyclase System". Excerpta Medica Int. Cong. Ser. (P.P. Foa, in press.